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The Human KLK8 (Neuropsin/Ovasin) Gene: Identification of Two Novel Splice Variants and Its Prognostic Value in Ovarian Cancer

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The Human KLK8 (Neuropsin/Ovasin) Gene: Identification of Two Novel Splice Variants and Its Prognostic Value in Ovarian Cancer 806 Vol. 7, 806–811, April 2001 Clinical Cancer Research The Human KLK8 (Neuropsin/Ovasin) Gene: Identification of Two Novel Splice Variants and Its Prognostic Value in Ovarian Cancer Angeliki Magklara, Andreas Scorilas, KLK8 encodes for a predicted secreted protein, its detection Dionyssios Katsaros, Marco Massobrio, in serum may aid in ovarian cancer diagnosis. George M. Yousef, Stefano Fracchioli, 1 Saverio Danese, and Eleftherios P. Diamandis INTRODUCTION Department of Pathology and Laboratory Medicine, Mount Sinai The human kallikrein gene family is a subfamily of serine Hospital and Department of Laboratory Medicine and Pathobiology, proteases, located at the chromosomal locus 19q13.3–q13.4. University of Toronto, Toronto, Ontario, M5G 1X5 Canada [A. M., A. S., G. M. Y., E. P. D.], and Departments of Obstetrics and Until recently, this family was known to include only three Gynecology, Gynecologic Oncology Unit [D. K., M. M., S. F.] and members: the pancreatic/renal kallikrein gene (KLK1), the hu- Gynecology, S. Anna Hospital [S. D.], University of Turin, Turin man glandular kallikrein 2 gene (KLK2), and prostate-specific 10126, Italy antigen (KLK3). In the past few years, another 11 kallikrein-like genes were discovered (reviewed in Ref. 1). Neuropsin is one of ABSTRACT these genes that maps to this locus (1, 2). According to the KLK8 (neuropsin/ovasin) is a new member of the human approved human kallikrein gene nomenclature, neuropsin is also kallikrein gene family, which consists of enzymes with serine known as KLK8 (3). protease enzymatic activity. Recent reports have implicated Neuropsin is a well-characterized, brain-related serine KLK8 in ovarian cancer. KLK8 may have potential clinical protease in mouse, where it has been shown to play an value for disease diagnosis or prognosis and it may also be a important role in neural plasticity (4), as well as in embryonic useful therapeutic target. cell differentiation (5). Recently, the human homologue of Purpose: We undertook this study to evaluate the prog- mouse neuropsin was cloned and found to be highly ex- nostic value of KLK8 in ovarian carcinoma by examining its pressed in brain, as well as in skin (6). Analysis of the expression in ovarian tumors. genomic organization of the human kallikrein 8 gene (KLK8) Experimental Design: The KLK8 gene was analyzed by revealed that it is composed of six exons and five introns, the reverse transcription-PCR and direct sequencing in several first exon being non-coding (6). The cDNA has a single open human normal tissues. Subsequently, its expression was reading frame of 780 bp encoding for a predicted 260-amino studied in a set of ovarian tumors, and statistical analysis acid protein. Two alternatively spliced forms of KLK8 have was performed. been identified so far (7, 8). The first (type 2 neuropsin) has Results: We have identified two novel mRNA splice an insert of 45 amino acids between the leader (pre)peptide variants of the KLK8 gene, which are abundantly expressed and the proenzyme (pro)peptide of the regular protein, be- in many tissues. These new variants were named KLK8 type cause of the presence of a second in-frame splice site in exon 3 and type 4. Study of the expression of the KLK8 gene and its spliced variants in ovarian tumors indicated that the new 3, which gives rise to a different mRNA form (7). The second variants were expressed very frequently and that full-length mRNA variant, known as tumor-associated differentially ex- KLK8 expression is an independent and favorable prognos- pressed gene-14, contains an additional new sequence of 491 Ј tic marker for ovarian cancer. Patients with higher KLK8 bp of 5 untranslated region; also, the nucleotides preceding expression in the tumor have lower grade disease, lower the poly(A) tail in the 3Ј untranslated region are not homol- residual tumor left after surgery, live longer, and relapse ogous with the regular form of neuropsin, but this mRNA less frequently. In multivariate analysis, higher KLK8 ex- form encodes for an identical protein as the human neuropsin pression was significantly associated with longer disease-free gene (8). Neuropsin/tumor-associated differentially ex- survival. pressed gene-14 was found to be overexpressed in ovarian Conclusions: These results suggest that KLK8 is a novel, carcinoma (8). In addition, a GenBank submission (accession favorable prognostic marker in ovarian cancer. Because number AF095742) refers to KLK8 as ovasin and indicates that it is a potential marker for ovarian cancer. In this report, we examine the expression of KLK8 in ovarian tumors by reverse transcription-PCR. While examining Received 9/11/00; revised 12/28/00; accepted 1/2/01. the expression of the gene in normal tissues, however, we found, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked along with the expected PCR band, two additional bands present advertisement in accordance with 18 U.S.C. Section 1734 solely to in all samples examined. Further analysis revealed that the two indicate this fact. bands represent novel, alternatively spliced forms of the KLK8 1 To whom requests for reprints should be addressed, at Department of gene. We here describe the prognostic value of the expression Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 Uni- versity Avenue, Toronto, Ontario, M5G 1X5 Canada. Phone: (416) 586- level of KLK8 and its two novel splice variants in a group of 143 8443; Fax: (416) 586-8628; E-mail: [email protected]. patients with ovarian cancer. Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2001 American Association for Cancer Research. Clinical Cancer Research 807 MATERIALS AND METHODS Table 1 Relationship between KLK8 (regular form) status and other Analysis of the KLK8 Gene by RT-PCR2 and Direct variables in 143 patients with primary ovarian cancer Sequencing. Total RNA isolated from 26 different human No. of patients (%) ␮ tissues was purchased from Clontech, Palo Alto, CA. Two gof KLK8 KLK8 RNA were reverse-transcribed into first-strand cDNA using the Patients negative positive P Superscript preamplification system (Life Technologies, Inc., KLK8 type 3 Gaithersburg, MD). The final volume was 20 ␮l. PCR was Negative 97 65 (67.0) 32 (33.0) 0.018a performed in a reaction mixture containing 1 ␮l of cDNA, 10 Positive 46 21 (45.7) 25 (54.3) mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl , 100 ␮M KLK8 type 4 2 a deoxynucleotide triphosphates, and 2.5 units of HotStar Taq Negative 47 33 (70.2) 14 (29.8) 0.11 Positive 96 53 (55.2) 43 (44.8) DNA polymerase (Qiagen, Mississauga, Ontario, Canada) in an Stage Eppendorf thermal cycler. The primers used were described I 27 12 (44.4) 15 (55.6) previously by Yoshida et al. (6), and their sequences were II 10 5 (50.0) 5 (50.0) 0.17b 5Ј-GTG-ACC-CCG-CCC-CTG-GAT-T-3Ј (forward) and 5Ј- III 90 59 (65.6) 31 (34.4) Ј IV 11 8 (72.7) 3 (27.3) GGG-AGA-TCT-AGT-GCT-TAT-CCT-3 (reverse). The cy- xc 5 cling conditions were 95°C for 15 min to activate the HotStar Grade Taq polymerase, followed by 40 cycles of 94°C for 30 s, 62°C GBd 10 2 (20.0) 8 (80.0) for 30 s, 72°C for 1 min, and a final extension step at 72°C for G1 17 8 (47.1) 9 (52.9) 0.007b 10 min. Equal amounts of PCR products were electrophoresed G2 23 12 (52.2) 11 (47.8) G3 89 62 (69.7) 27 (30.3) on 1% agarose gels and visualized by ethidium bromide stain- x4 ing. The three visible bands were cut out and purified using the Histotype Gel Purification kit (Qiagen), and the purified DNA was directly Serous 63 37 (58.7) 26 (41.3) b sequenced. To ensure that the first (non-coding) exon is in- Endometrioid 25 13 (52.0) 12 (48.0) 0.32 Undifferentiated 22 17 (77.3) 5 (22.7) cluded in the variant mRNAs, we constructed a new forward Others 31 18 (58.1) 13 (41.9) primer (5Ј-GTT-CCC-AGA-AGC-TCC-CCA-G-3Ј), binding at x2 the beginning of exon 1, and performed new PCR analysis using Residual tumor (cm) the aforementioned reverse primer. 0 56 27 (48.2) 29 (51.8) b Patients with Ovarian Cancer. Included in this study 1–2 27 16 (59.3) 11 (40.7) 0.044 Ͼ2 53 38 (71.7) 15 (28.3) were 143 consecutive patients with epithelial ovarian carci- x7 noma, with ages ranging from 25 to 80 with a median of 58 Menopause years. Patients underwent surgery and treatment for primary Pre/peri 49 27 (55.1) 22 (44.9) 0.47a ovarian carcinoma at the Department of Obstetrics and Gyne- Post 94 59 (62.8) 35 (37.2) Response to CTXe cology, Gynecological Oncology Unit, University of Turin, NC/PD 15 10 (66.7) 5 (33.3) 0.59a between 1991 and 1999. Follow-up information (median fol- CR/PR 118 69 (58.5) 49 (41.5) low-up period, 48 months) was available for 135 patients, NE 10 among whom 81 (60%) had relapsed and 57 (42%) died. Pa- a ␹2 test. tients were staged according to the criteria of the International b Fisher’s exact test. Federation of Gynecology and Obstetrics (9); information was c x, unknown status. d available for 138 of them (Table 1). Specifically, 27 (20%) GB, borderline grade. e CTX, chemotherapy; NC, no change; PD, progressive disease; patients had stage I disease, 10 (7%) had stage II, 90 (65%) had CR, complete response; PR, partial response; NE, not evaluated.
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